Optimization Of Fermentation Conditions For Bacillus Licheniformis W10Producing Antifungal Protein And Its Application Techniques

Abstract:Bacillus licheniformis W10is an antagonistic bacterium that has significant inhibition effect on Botrytis cinerea and other important plant pathogens. Bacillus licheniformis W10can produce antifungal protein, which can evidently damage the mycelial cell, and inhibit the pathogen growth and sporulation. The experiments of optimizing the fermentation medium and conditions of the strain W10were carried out. Furthermore, this paper studied the application techniques of the antifungal protein.The results of the experiments concluded that the optimum fermentation medium of strain W10consisted of10.131g/L soybean flour,1.126g/L peptone,16.894g/L corn flour,2.413g/L glucose,7.965g/L ammonium sulfate,0.45g/L K2HPO4,0.45g/L MgSO4, and4.5g/L NaCl.Also, the optimum fermentation conditions were12h of seed cell age;4days of fermentation time;2%inoculation volume of seed bacterium; pH vaule of7.0; dissolved oxygen value of0-20h30%and20-60h20%. The fermentation method used was fed-batch culture with10g/L glucose and5g/L peptone as supplement medium. The supplement medium volume was500mL. The first time feeding the supplement medium was at1Oh after the fermentation start and the second time was at18h. The bubble enemy was selected to be the optimum defoamer to eliminate bubbles. The desired concentration was200mg/L and the volume used was100mL.Under the optimum conditions, the inhibitory effect of the strain W10fermented using the best culture media on Botrytis cinerea reached99.5%, showing a34.6%increase compared to73.9%before optimization.The different application methods of the antifungal protein had a great impact on the disease control effect on Botrytis cinerea. To measure the synergistic effects, the antifungal protein was mixed with procymidone and boscalid, respectively, in the experiments. The results showed that the synergistic effects were produced when the ratios of the antifungal protein to procymidone in the mixture were1:1and1:5. The synergistic coefficients were230.4114and141.0645, respectively. In contrast, the synergistic effects of the protein-boscalid mixture were not observed.Under the control, using the antifungal protein before the pathogen invaded its host, the control efficiency could get up to73.72. However, at the early stage of the disease, the control efficiency was66.38%, and after the disease spots extending, the control efficiency was only46.72%. Improving the concentration of the antifungal protein could enhance control effect. When the dose of the protein were1mg/mL,2mg/mL and3mg/mL, the control efficiencies were respectively73.57%,81.82%and86.79%.Increasing the number of times of the antifungal protein application was also beneficial to the diseases control. When it was used once, the control efficiency was74.43%. When it was used twice, the efficiency was83.49%, and with three times, the efficiency was88.41%. Moreover, using the antifungal protein under different temperatures, the control effect was undifferentiated.The study of disease control of tomato grey mould using the antifungal protein was also carried out. In10days after applying antifungal protein, the disease control effects on leaves necrotic spot with the dosages of25g/mu and50g/mu were66.7%and68.3%, respectively. The control effects after20days remained at60.61%and65.34%, which were still higher than the effect of procymidone with25g/mu dosage (45.3%). After removing all diseased fruit before using agentia, there was no disease fruits appearing in the test field using both the antifungal protein and procymidone after10days. However, in contrast the disease fruit rate tested by control was5.6%.